Ruva Is a Sliding Collar That Protects Holliday Junctions from Unwinding While Promoting Branch Migration

doi:10.1016/j.jmb.2005.10.075 J. Mol. Biol. (2006) 355, 473–490

RuvA is a Sliding Collar that Protects Holliday Junctions from Unwinding while Promoting Branch Migration

Daniel L. Kaplan1* and Mike O’Donnell1,2

1Rockefeller University, The RuvAB proteins catalyze branch migration of Holliday junctions Laboratory of DNA Replication during DNA recombination in Escherichia coli. RuvA binds tightly to the New York, NY 10021, USA Holliday junction, and then recruits two RuvB pumps to power branch migration. Previous investigations have studied RuvA in conjunction with 2Howard Hughes Medical its cellular partner RuvB. The replication fork helicase DnaB catalyzes Institute, Laboratory of DNA branch migration like RuvB but, unlike RuvB, is not dependent on RuvA Replication, New York, NY for activity. In this study, we specifically analyze the function of RuvA by 10021, USA studying RuvA in conjunction with DnaB, a DNA pump that does not work with RuvA in the cell. Thus, we use DnaB as a tool to dissect RuvA function from RuvB. We find that RuvA does not inhibit DnaB-catalyzed branch migration of a homologous junction, even at high concentrations of RuvA. Hence, specific protein–protein interaction is not required for RuvA mobilization during branch migration, in contrast to previous proposals. However, low concentrations of RuvA block DnaB unwinding at a Holliday junction. RuvA even blocks DnaB-catalyzed unwinding when two DnaB rings are acting in concert on opposite sides of the junction. These findings indicate that RuvA is intrinsically mobile at a Holliday junction when the DNA is undergoing branch migration, but RuvA is immobile at the same junction during DNA unwinding. We present evidence that suggests that RuvA can slide along a Holliday junction structure during DnaB-catalyzed branch migration, but not during unwinding. Thus, RuvA may act as a sliding collar at Holliday junctions, promoting DNA branch migration activity while blocking other DNA remodeling activities. Finally, we show that RuvA is less mobile at a heterologous junction compared to a homologous junction, as two opposing DnaB pumps are required to mobilize RuvA over heterologous DNA. q 2005 Elsevier Ltd. All rights reserved. Keywords: DNA replication; DNA recombination; Holliday junction; branch *Corresponding author migration; RuvA

Introduction which is processed by the RuvABC proteins.2 The RuvA protein initially binds the Holliday junction, DNA recombination functions in Escherichia coli and then recruits RuvB protein rings to opposite torepairdamagedDNAandrescuestalled sides of the junction.3,4 RuvB is a molecular motor replication forks.1 During recombination, a DNA that uses the energy derived from ATP binding and strand is paired with its homolog from a different hydrolysis to drive unidirectional movement of the duplex in a reaction catalyzed by RecA working in four-way junction.5 The RuvAB complex then concert with other proteins.1 This creates a four- recruits RuvC to the junction.6 RuvC is a nuclease way DNA structure, also called a Holliday junction, that cleaves the Holliday junction, thereby resolving it into two duplex DNAs.7 RuvAB has been studied by biochemical and Present address: D. L. Kaplan, Vanderbilt University, Department of Biological Sciences, Nashville, TN 37232, structural techniques. RuvA binds as a tetramer or USA. octamer to a Holliday junction. The RuvA protein Abbreviation used: ssDNA, single-stranded DNA. has acidic pins that inhibit binding to double- E-mail address of the corresponding author: stranded DNA, thereby targeting the protein to 8–11 [email protected] Holliday junctions. RuvB is a hexameric ring

0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. 474 RuvA Promotes Branch Migration protein with a central channel wide enough to mobilizes RuvA using only RuvA and RuvB, since encircle double-stranded DNA.11–13 RuvB alone the activities of these two proteins are dependent does not bind Holliday junction DNA under upon one another.2 Unlike RuvB, DnaB does not physiological conditions, but after RuvA binds the require RuvA for activation. Thus, we investigated Holliday junction, RuvA facilitates the assembly of how RuvA moves at a Holliday junction by using two RuvB rings onto opposite sides of the RuvA- DnaB in conjunction with RuvA. In this study, DnaB junction (Figure 4(a)).14–16 The two RuvB rings are is used as a tool to study how RuvA functions, since thought to function in concert as DNA pumps that these two proteins do not function together in vivo. drive branch migration of the Holliday junction.15 There is an additional advantage to using DnaB DnaB functions in DNA replication and is a with RuvA to study RuvA function. RuvB rings member of the class of ring-shaped helicases.17–19 bind to opposite arms of a RuvA-bound Holliday DnaB is the primary replicative helicase of E. coli, junction in either of two orientations (Figure 4(a)). and unwinds the parental duplex to provide single- Thus, it is difficult to target RuvB loading to a stranded DNA (ssDNA) for the replicative poly- particular junction arm. In contrast, DnaB loads merases.20 The DnaB hexamer encircles ssDNA onto junction-arm DNA only if a 50single-strand while translocating along it, pumping the strand extension (50 tail) is attached to a particular junction through the central channel.21–24 Upon encounte- arm. Thus, unlike RuvB, one DnaB hexamer can be ring a forked duplex structure, the second DNA loaded onto a particular junction arm by specifically strand cannot fit into the central channel of DnaB, adding a 50 loading tail. and therefore the continued advance of DnaB along We find that RuvA binds tightly to Holliday the original strand forces its separation from the junction DNA and blocks DnaB-catalyzed unwin- second DNA strand.24,25 It is thought that DnaB, ding of a Holliday junction. Unwinding activity is like other hexameric helicases, may act at the blocked even when two DnaB hexamers act in replication fork to unwind parental DNA in this concert. However, RuvA does not block DnaB- manner.26–28 catalyzed branch migration of a homologous Holli- The DnaB hexamer can also operate in a mode in day junction. Hence, RuvA does not need specific which it encircles both strands of duplex DNA. In protein activation by RuvB to mobilize in the this mode, the DnaB does not unwind the DNA.24 direction of branch migration. We present evidence However, DnaB actively translocates along the that suggests that RuvA slides along the Holliday duplex while encircling two DNA strands as it junction during DNA branch migration, but not powers branch migration of Holliday junctions.29 during DNA unwinding. Interestingly, two DnaB Although this reaction is very efficient in vitro, the pumps are needed to power migration of RuvA in vivo role for DnaB-catalyzed branch migration is over heterologous DNA, an action that fits nicely unclear. In summary, DnaB unwinds DNA when with the physiological architecture of two RuvB encircling one DNA strand, and drives DNA branch pumps straddling the RuvA protein at a Holliday migration while encircling two DNA strands. junction. DnaB and RuvB have several mechanistic features in common. For example, DnaB and RuvB are both hexameric ring proteins that encircle Results DNA.2 Furthermore, both DnaB and RuvB utilize ATP binding and hydrolysis to unwind DNA with Homologous and heterologous Holliday 50 to 30 polarity.24,30–32 DnaB and RuvB also displace junction DNA substrates used in this study proteins bound to DNA,29,33 and they both drive branch migration of Holliday junctions.15,29 Finally, Holliday junctions in the cell are usually homo- two DnaB rings can bind to opposite sides of a logous, as RecA normally pairs DNA strands of the Holliday junction, and work in concert to drive same sequence to create the junction. By homolo- branch migration of an extended heterologous gous, we mean that the DNA arms contain junction, like RuvB.34–36 complementary sequences before and after branch Biochemical studies of RuvA in the past have migration. Figure 1(a) shows a homologous Holli- been performed with RuvA in conjunction with its day junction substrate used in this study. Note that cellular partner, RuvB. The biochemical action of the substrate is not completely homologous, other- the RuvAB complex is thus well studied. However, wise the Holliday junction is unstable and can some intrinsic properties of RuvA during its action migrate spontaneously. Thus, the homologous are unclear, as it is most often studied in conjunction substrate used here bears a small degree of with RuvB. This leaves unanswered a number of heterology (5 bp) to stabilize the structure and questions of how RuvA functions. For example, render it amenable to experimentation. In the how does RuvA bind tightly to Holliday junctions, reaction shown in Figure 1(a), the Holliday junction yet become activated to move during branch branch-point migrates a distance of 45 bp, of which migration? Previous proposals suggest that RuvB 40 are complementary, while five are non-comple- must mobilize RuvA bound to a Holliday junction, mentary. and that this action is mediated via specific protein– Heterologous junctions contain DNA sequence protein interaction between RuvB and RuvA.10 that is non-complementary, and the DNA arm will However, it is difficult to study the process that therefore become unpaired after branch migration RuvA Promotes Branch Migration 475

Figure 1. Homologous and heterologous Holliday junction DNA substrates used in this study. The substrates shown are used in this study to assess RuvA mobility at (a) a homologous and (b) heterologous Holliday junction. (a) This homologous substrate is used in Figure 2(a). The 1-2 and 3-4 duplexes are 45 bp in length, and the 1-4 and 2-3 duplexes are 25 bp in length. The 50 tail is composed of 30 dT residues. Oligonucleotides used to form this substrate are provided in Table 1. In the reaction shown, the branch-point migrates for 45 bp, of which 40 bp are complementary, and 5 bp are non-complementary. (b) This heterologous substrate is used in Figure 6(a). The 1-2 and 3-4 duplexes are 25 bp in length, and the 1-4 and 2-3 duplexes are 25 bp in length. The 50 tail is composed of 30 dT residues. In the reaction shown, the branch-point migrates over 19 non-complementary base-pairs. over the heterologous region. Heterologous Holli- homologous junction for 0.5 min to 4 min at 37 8C. day junctions may be found in the cell during The homologous junction is composed of strands 1, episodes of DNA damage, when the integrity and 2, 3, and 4. Strand 1 is radiolabeled (asterisk, strand sequence fidelity of the DNA are compromised. *1). Reactions were quenched and products were Thus, the cell may contain heterologous Holliday resolved on a native gel, which separates the larger junctions under conditions of DNA damage. In this junction substrate from the smaller branch study, the mobility of RuvA at homologous migration product that migrates faster. DnaB and heterologous DNA structures are investigated, promotes substantial branch migration of this since either may occur in vivo. Figure 1(b) shows a substrate, as expected (Figure 1(a), accumulation heterologous junction substrate used in of *1-4 duplex). The model above each gel is used to this study. For this substrate, 19 bp of contiguous, orient the reader, and was determined from the non-complementary DNA sequence are present in experimental gel evidence below. The arrows next the DNA products after branch migration. to the gel show the migration distance of DNA products as determined by radiolabeled DNA RuvA inhibits DnaB-catalyzed unwinding, but markers that were analyzed in the same gel in the not DnaB-catalyzed branch migration, of a experiments described here. For clarity, however, homologous junction markers are cropped from the gel images. In Figure 2(b), the substrate was first incubated Here, we use DnaB as a tool to study how RuvA with RuvA for 1 min, followed by addition of DnaB functions at a Holliday junction. In the experiment as in Figure 2(a). Nearly identical results were illustrated by Figure 2(a), DnaB was incubated with obtained in the absence or in the presence of RuvA a long homologous Holliday junction with a single (compare Figure 2(a) with (b), and view quantifi- 50 loading tail for DnaB (also illustrated in cation of product accumulation in Figure 2(c)). Figure 1(a)). We have shown that with only a 50 Therefore, RuvA does not inhibit DnaB-catalyzed tail and no 30 tail, DnaB tracks on the ssDNA tail branch migration of a homologous junction. This and then, instead of unwinding, it travels onto the result suggests that RuvA is intrinsically mobile on duplex by encircling both strands and catalyzes a homologous Holliday junction during DNA branch migration.35 DnaB was incubated with the branch migration, and does not require activation 476 RuvA Promotes Branch Migration

Figure 2. RuvA inhibits DnaB-catalyzed unwinding, but not DnaB-catalyzed branch migration, of a homologous junction. The model above each gel is used to orient the reader, and was determined from the experimental gel evidence below. (a) DnaB acts on a long homologous Holliday junction with one 50 tail. The 1-2 and 3-4 duplexes are 45 bp in length, and the 1-4 and 2-3 duplexes are 25 bp in length. The 50 tail is composed of 30 dT residues. Oligonucleotides used to form this substrate are provided in Table 1. DnaB was incubated with the junction illustrated for the time indicated. Native gel analysis of the reaction is shown. The arrows next to the gel show the migration distance of DNA products as determined by radiolabeled DNA markers electrophoresed in the same gel. Markers are cropped from the gel images for clarity. (b) Same as (a), except the substrate is pre-incubated with RuvA for 1 min prior to adding DnaB. (c) Quantification of the accumulation of branch migration product (*1, 4) from (a) and (a). (d) DnaB acts on a long homologous Holliday junction with one fork. DnaB was incubated with the junction DNA illustrated for the time indicated. The substrate is identical with (a), except strand 4 contains a 27 bp double-stranded 30 tail. Native gel analysis of the reaction is shown. (e) Same as (d), except the substrate is pre-incubated with RuvA for 1 min prior to adding DnaB. (f) Quantification of the accumulation of unwinding products (*2, 3, 4 and *2) from (a) and (b). through specific protein–protein contacts with forked.29 DnaB loads onto the 50 tail of this forked RuvB. substrate. The 30 tail of the fork is double-stranded, The previous experiment demonstrates that which promotes DnaB-catalyzed unwinding by RuvA does not inhibit DnaB-catalyzed branch aiding exclusion of this 30 tail from the central migration of a homologous Holliday junction. channel of DnaB.24 DnaB first unwinds strand 1 of Does RuvA inhibit other DNA-modulating this substrate to produce an intermediate product reactions at a Holliday junction? To test if RuvA composed of strand *2, strand 3, and the 4 duplex inhibits other DNA remodeling activities, RuvA (see reaction (i) in Figure 2(d), and the gel band at and DnaB were incubated with a homologous early time-points that corresponds to this inter- Holliday junction bearing a forked structure at mediate product). Later in the reaction, DnaB one end (Figure 2(d)). We have shown that DnaB unwinds strand *2 of this intermediate product to will unwind a Holliday junction if the loading site is yield free strand *2 (see reaction (ii) in Figure 2(d), RuvA Promotes Branch Migration 477 and the gel band at later time-points corresponding branch migration of this substrate, as observed to free strand *2). (Strand 2 of this substrate is previously.29 RuvA blocks DnaB-catalyzed unwin- labeled instead of strand 1 to definitively distin- ding of this substrate, but it does not inhibit guish unwinding from branch migration. If strand 1 DnaB-catalyzed branch migration of this substrate. were labeled here, DnaB could unwind the 1-4 Thus, RuvA inhibits DnaB unwinding activity, but product to yield free strand 1, thereby making it not branch migration, at a homologous junction. difficult to distinguish branch migration from These results suggest that RuvA is free to move at a unwinding. The DnaB enzyme is not fully trapped Holliday junction during DNA branch migration, in these reactions, and it may react with the primary while RuvA is immobile at the same Holliday products generated to produce secondary reaction junction during DNA unwinding. products.) We next performed several control experiments This experiment was then repeated, but the to test if our RuvAB proteins behave as described by substrate was first incubated with RuvA other laboratories. RuvA binds to Holliday junc- (Figure 2(e)). The result shows that RuvA inhibits tions, and then recruits two RuvB hexameric rings the accumulation of unwinding products substan- to opposite sides of RuvA. The RuvAB complex tially (compare Figure 2(d) with (e) and see the then pumps DNA to drive branch migration. To test quantification of unwinding product accumulation if our RuvA and RuvB proteins are able to drive in Figure 2(f)). Thus, RuvA inhibits DnaB-catalyzed branch migration, as others have reported, the two unwinding of this homologous Holliday junction proteins were incubated with a radiolabeled hetero- substrate. This observation suggests that RuvA logous Holliday junction (Figure 4(a)). Branch blocks DNA unwinding at a Holliday junction. migration of this heterologous junction substrate In Figure 3, DnaB was incubated with a Holliday will result in two smaller products (Figure 4(a)). junction that is similar to that of Figure 2(d), except RuvA was incubated with the heterologous junction the 30 tail is single-stranded instead of double- for 1 min at 37 8C, followed by RuvB for 0.5 min to stranded. With a single-stranded 30 tail, DnaB now 4 min. RuvB rings usually encircle the *1-2 and 3-4 has a similar probability of catalyzing unwinding or duplexes to drive branch migration of this

Figure 3. RuvA inhibits DnaB- catalyzed unwinding, but not DnaB-catalyzed branch migration, of a homologous junction at a single DNA substrate. The model above each gel is used to orient the reader, and was determined from the experimental gel evidence below. (a) DnaB acts on a long homologous Holliday junction with one fork. The 1-2 and 3-4 duplexes are 45 bp in length, and the 1-4 and 2-3 duplexes are 25 bp in length. Each 50 tail is composed of 30 dT, and each 30 tail is composed of 30 dT. Oligonucleo- tides used to form this substrate are provided in Table 1. DnaB was incubated with the junction illustrated for the time indicated. Native gel analysis of the reaction is shown. The arrows next to the gel show the migration distance of DNA products as determined by radiolabeled DNA markers electrophoresed in the same gel. Markers are cropped from the gel images for clarity. (b) Same as (a), except the substrate is pre-incubated with RuvA for 1 min prior to adding DnaB. 478 RuvA Promotes Branch Migration

Figure 4. RuvA does not recruit DnaB to Holliday junctions. The model above each gel is used to orient the reader, and was determined from the experimental gel evidence below. (a) RuvAB activity on a heterologous Holliday junction. The numbers represent names for each DNA strand. Strand 1 is labeled with 32P at the 50 terminus (asterisk). Duplexes 1-2 and 3-4 are 45 bp in length, and duplexes 1-4 and 2-3 are 25 bp in length. Oligo- nucleotides used to form this substrate are provided in Table 1. RuvA was incubated with the heterologous junction illustrated for 1 min, followed by incubation with RuvB for the periods of time indicated. Native gel analysis of the reaction products is shown. The arrows next to the gel show the migration distance of DNA products as determined by radiolabeled DNA markers electrophoresed in the same gel. Markers are cropped from the gel images for clarity. (b) Same as (a), except RuvA is omitted from the reaction. (c) DnaB acts on the heterologous Holliday junction illustrated in (a). DnaB was incubated with the heterologous junction illustrated for the time periods indicated. Native gel analysis of the reaction is shown. (d) Same as (c), except the substrate is pre-incubated with RuvA for 1 min prior to adding DnaB. substrate, producing the *1-2 product. This reaction protein (not shown). In summary, the RuvAB is shown in scheme (i) of Figure 4(a), and confirmed proteins used in this study drive branch migration by the dark product band in the gel. Less frequently, of Holliday junctions as expected from the work of RuvB encircles the *1-4 and 2-3 duplexes to drive other laboratories.8,15,34,37 branch migration, producing the radiolabeled *1-4 Next, we performed an additional control to duplex product (scheme (ii) of Figure 4(a), and faint ensure that RuvA does not influence DnaB loading branch migration product in the gel). onto a Holliday junction. We do not expect RuvA to Reaction (i) of Figure 4(a) occurs far more load DnaB, as there is no genetic evidence to frequently than reaction (ii). The reaction preference suggest that RuvA recruits DnaB. However, we correlates with the length of the duplex arms. This performed the following control experiment just to heterologous junction has arm lengths of 45 bp for be sure. DnaB was incubated with the Holliday duplexes 1-2 and 3-4, and 25 bp for duplexes 1-4 junction lacking a 50 tail for 0.5 min to 4 min and 2-3. The binding site size for RuvAB is (Figure 4(c)). Very little product accumulation is approximately 35 bp on either side of the junction.15 observed (Figure 4(c)), as expected for a substrate Thus, there is likely competition between these two with no 50 tail. To determine if RuvA recruits DnaB reactions, and the longer duplex arms act as better to a Holliday junction, RuvA was incubated with binding surfaces for the RuvB rings. In the absence the junction DNA for 1 min at 37 8C, then DnaB was of RuvA, RuvB alone produces very little product added to the reaction for 0.5 min to 4 min (Figure 4(b)). RuvA alone does not produce any (Figure 4(d)). There is no conversion of substrate product (not shown), and the substrate does not to product during this time-frame (Figure 4(d)). disassemble spontaneously in the absence of Thus, RuvA does not load DnaB at a Holliday RuvA Promotes Branch Migration 479 junction, as expected for this control. These results published studies, and may correspond to tetra- allow one to conclude that in Figures 2 and 3, DnaB meric RuvA, octameric RuvA, and higher-order self-loads on the 50 single-stranded region of the species.16) Quantification of the results of this gel is substrate in a manner independent of the presence shown in Figure 5(c). RuvA binds half the junction of RuvA. DNA at a protein concentration of approximately How does RuvA inhibit DnaB unwinding activity 10 nM. Thus, RuvA binds tightly to Holliday at a Holliday junction in Figures 2 and 3? One junction DNA under our reaction conditions, and explanation is that RuvA binds tightly to the this provides a simple explanation for how RuvA junction substrate, and creates a physical block to inhibits DnaB unwinding activity. DnaB movement. In support of this idea, it has been Tight binding of RuvA to Holliday junctions and demonstrated that RuvA binds tightly to Holliday the resulting inhibition of unwinding by DnaB raise junctions.16 To test if RuvA binds to junction DNA an interesting question. Why does RuvA not block under our reaction conditions, an electrophoretic DnaB branch migration activity? One possible mobility-shift assay (EMSA) was performed. Radio- explanation is that DnaB readily dislodges, or labeled junction DNA was incubated with RuvA disassembles, the RuvA multimer from the junction under conditions that are identical with those used DNA during branch migration but not unwinding. in our branch migration and unwinding assays If this is the case, then DnaB must be far more active (Figure 5(a)). The sample was then analyzed by in dislodging RuvA when DnaB encircles two DNA electrophoreses in a native polyacrylamide gel to strands during branch migration compared to when separate the RuvA–DNA complex from free DNA. DnaB encircles one strand during unwinding. As the concentration of RuvA is increased, bands However, previous studies make this hypothesis corresponding to RuvA–DNA complexes increase unlikely. The rate of DnaB-catalyzed protein in intensity (Figure 5(b)). (The appearance of several displacement from DNA is similar for DnaB RuvA–junction DNA complexes is consistent with encircling two DNA strands and for DnaB encircling one DNA strand.29 In addition, DnaB is quite slow to displace tightly bound proteins from DNA relative to the rate of DnaB-catalyzed branch migration activity.29 An alternative hypothesis to account for the observation that RuvA blocks DnaB-catalyzed unwinding but not branch migration is that RuvA can slide along the Holliday junction structure during branch migration, but not unwinding (Figure 10). For DnaB to catalyze unwinding of an RuvA-bound Holliday junction, RuvA is probably displaced, since the four-way junction structure is destroyed during the reaction (Figure 10(a)). In contrast, during DnaB-catalyzed branch migration of an RuvA-homologous junction complex, the junction structure is preserved continuously (until the junction is eventually split when branch migration reaches the end of the substrate). This may enable RuvA to stay bound to DNA throughout the branch migration process (Figure 10(b)). This proposed difference in mechanism, in which RuvA slides during branch migration but is displaced directly off a junction during unwinding may explain the difference in RuvA blocking ability. In other words, RuvA binds Holliday junctions stably, and it may be difficult to dislodge this protein from an internal position, but during branch migration RuvA may slide along with the junction. When branch migration is complete and the junction is split in two, then RuvA would presumably slide off the end of the DNA. We would like to determine directly whether Figure 5. RuvA binds tightly to Holliday junctions. RuvA is displaced from DNA or slides along the Electrophoretic mobility-shift analysis of RuvA binding a junction structure during DnaB-catalyzed branch Holliday junction. (a) Scheme for RuvA binding a heterologous junction. The duplex arm lengths are each migration. However, this cannot be determined by 25 bp. (b) After RuvA incubation for 1 min with the examining whether RuvA is on or off DNA, as radiolabeled junction illustrated in (a), the reaction is RuvA must fall off the DNA substrate in both analyzed by native gel electrophoresis. (c) The data from scenarios, whether it is dislodged from the internal (b) are quantified and plotted. junction structure directly, or whether it slides off 480 RuvA Promotes Branch Migration the end only after branch migration is complete. In RuvA inhibits branch migration of both cases, RuvA is bound to DNA only transiently. a heterologous junction catalyzed Furthermore, whether DnaB dislodges RuvA by a single DnaB ring directly, or DnaB slides with RuvA, DnaB and RuvA will be bound to the Holliday junction at the In the experiments of Figure 6 we examine RuvA same time when DnaB loads onto the RuvA-bound and DnaB on a heterologous junction substrate to Holliday junction. Thus, simply showing that DnaB help distinguish between the sliding and direct and RuvA are bound to junction DNA at the same displacement models for branch migration time will not distinguish between RuvA sliding and presented above. Studying RuvA action at a RuvA displacement. We next present an indirect heterologous junction also has important biological method to examine whether RuvA is displaced or relevance, since RuvA is likely to encounter regions slides along the junction during DNA branch of heterology during DNA damage. The junction migration. substrate of Figure 6(a)–(c) is similar to that of

Figure 6. RuvA inhibits branch migration of a heterologous junction catalyzed by a single DnaB ring. The model above each gel is used to orient the reader, and was determined from the experimental gel evidence below. (a) DnaB acts on a short heterologous Holliday junction with one 50 tail. Each duplex is 25 bp in length, and the 50 tail is composed of 30 dT. Oligonucleotides used to form this substrate are provided in Table 1. DnaB was incubated with the junction illustrated for the periods of time indicated. Native gel analysis of the reaction is shown. The arrows next to the gel show the migration distance of DNA products as determined by radiolabeled DNA markers electrophoresed in the same gel. Markers are cropped from the gel images for clarity. (b) Same as (a), except the substrate is pre-incubated with RuvA for 1 min prior to adding DnaB. (c) Data from gels shown in (a) and (b) were quantified and plotted. The products that are quantified in (c) are all derived from the *1-4 branch migration product. (d) Filled triangles show data from an experiment similar to that shown in (b), except the concentration of RuvA is varied and the time of DnaB incubation is fixed at 2 min. Open circles show data from a similar experiment using the substrate from Figure 2(b) (4 min DnaB). DnaB incubation times were chosen to roughly match product accumulation in the absence of RuvA. (e) RuvA and RuvB acting on the junction illustrated in (a). RuvA was incubated with the junction illustrated for 1 min, followed by incubation with RuvB for the periods of time indicated. (f) Same as (e), except RuvA is omitted from the reaction. RuvA Promotes Branch Migration 481

Figures 2(a)–(c), except the heterologous junction of with strand 4 and slows the rate of strand Figure 6(a)–(c) will yield unannealed duplex arms annealing.29) The additional faint bands in once branch migration is complete, unlike the Figure 6(a) arise from DnaB-catalyzed unwinding homologous junction of Figure 2(a)–(c). The core of the branch migration product, followed by strand region of the junction has an identical DNA reannealing.29 sequence for the substrates used in Figures 2–6. The effect of RuvA on DnaB-catalyzed branch Thus, the DNA-binding site for RuvA is similar in migration of the heterologous junction is shown in all of these experiments. We have shown elsewhere Figure 6(b). The result shows that the branch that with the junction in Figure 6(a)–(c), DnaB loads migration product is produced far more slowly in onto strand 1 and travels onto the duplex by the presence of RuvA (compare Figure 6(a) with (b)). encircling strands 1 and 4. DnaB then catalyzes Quantification of all products produced in Figure 6(a) branch migration while encircling strands 1 and 4.29 and (b) shows that RuvA substantially inhibits DnaB DnaB encircles two DNA strands and pumps DNA activity at this heterologous junction (Figure 6(c)). in a similar manner in the junction of Figure 6(a)–(c) Thus, RuvA inhibits DnaB-catalyzed branch and that of Figure 2(a)–(c). Thus, the ability of DnaB migration at the heterologous junction of Figure 6. to dislodge RuvA from the junction of The concentration-dependence of RuvA inhibition Figure 6(a)–(c) should be the same as the ability of of DnaB activity on this heterologous junction was DnaB to dislodge RuvA from the junction of studied (Figure 6(d), filled triangles). There is Figure 2(a)–(c). Hence, if DnaB displaces RuvA substantial inhibition of DnaB-catalyzed branch rapidly during branch migration on a homologous migration over a wide range of concentrations of junction, RuvA should also have little effect on RuvA. This result contrasts markedly with a similar migration of a heterologous junction, since DnaB experiment performed with the homologous junction should simply knock it off the DNA and then substrate from Figure 1(a) (Figure 6(d), open circles), proceed on its way. where RuvA does not inhibit DnaB over the same A different result maybe expected if RuvA slides range of concentrations. Thus, RuvA blocks DnaB- during branch migration. Once branch migration catalyzed branch migration of a heterologous junc- starts, the DNA strands in the junction of Figure 6(a)– tion, but not of a homologous junction. If DnaB (c) will become non-complementary, unlike in dislodges RuvA from the homologous substrate, it Figure 2(a)–(c). Thus, RuvA may be expected to should also dislodge RuvA rapidly from the heter- have difficulty sliding over this heterologous junction ologous substrate. However, RuvA inhibits DnaB once branch migration starts, because the junction greatly on a heterologous junction. Thus, the rapid structure will become distorted (see Figure 10(c)). rate of branch migration of the homologous junction RuvA is structurally suited to bind to a four-way in the presence of RuvA supports the model whereby junction of annealed, duplex DNA.10 If the arms RuvA slides over the homologous Holliday junction become non-complementary,RuvAwill not be able to when DnaB pushes it (Figure 10(a)). optimally accommodate the unannealed duplex As an additional control, we tested RuvAB on the arms. Forcing unannealed arms through the RuvA heterologous junction, which should result in sandwich may be energetically unfavorable. The branch migration, as the work of other laboratories structures of the homologous and heterologous has shown.8,15,34,37 The results demonstrate that junctions differ from each other only after branch RuvAB is active on this substrate, with roughly migration has begun. Hence, if RuvA behaves equal accumulation of each branch migration differently at a heterologous junction compared to a product (Figure 6(e)). Each duplex arm of this homologous junction, then RuvA is likely recogniz- substrate has a length of 25 bp. The roughly equal ing the change in junction structure that arises only accumulation of each branch migration product once branch migration starts. In other words, if RuvA correlates with the equal size of the duplex arms. blocks DnaB at a heterologous but not a homologous Thus, the hypothesis that the length of the arms sets junction, then RuvA is likely bound to the junction up a competition between the two branch migration continuously during branch migration (i.e. RuvA reactions is supported by these data (i.e. results in slides). Thus, study of the effect of RuvA on DnaB Figure 4). There is very little activity when RuvA is using the heterologous substrate of Figure 6(a)–(c) excluded from the reaction (Figure 6(f)). may provide indirect evidence of how RuvA acts during DnaB-catalyzed branch migration of a hom- RuvA inhibits unwinding of a Holliday junction ologous junction (i.e. is RuvA displaced or does it catalyzed by a single DnaB ring slide?). First, we examined the results of DnaB action on We next studied the mobility of RuvA at heter- the heterologous junction in the absence of RuvA as ologous junctions during DNA unwinding. We a control (Figure 6(a)). DnaB promotes substantial incubated DnaB with a heterologous junction bearing accumulation of the *1-4 branch migration product, both 50 and 30 tails (a fork) (Figure 7(a)). The result as expected. The *1-4 duplex is a branch migration shows that DnaB unwinds the strand bearing the 50 product, and not the secondary result of strand tail from the rest of the junction (Figure 7(a), scheme reannealing. (The half-time of strand 1 annealing (i)). We have shown elsewhere that DnaB drives with strand 4 is 70 min, due to an intramolecular branch migration of this substrate (Figure 7(a), hairpin within the region of strand 1 that base-pairs scheme (ii)),29 and this product is observed, as 482 RuvA Promotes Branch Migration

Figure 7. RuvA inhibits unwinding of a Holliday junction catalyzed by a single DnaB ring. The model above each gel is used to orient the reader, and was determined from the experimental gel evidence below. (a) DnaB acts on a heterologous Holliday junction with one fork. Each duplex is 25 bp in length, and the 50 and 30 tails are each composed of 30 dT. Oligonucleotides used to form this substrate are provided in Table 1. DnaB was incubated with the junction illustrated for the periods of time indicated. Native gel analysis of the reaction is shown. The arrows next to the gel show the migration distance of DNA products as determined by radiolabeled DNA markers electrophoresed in the same gel. Markers are cropped from the gel images for clarity. (b) Same as (a), except the substrate is incubated with RuvA for 1 min before adding DnaB. (c) Data from gels shown in (a) and (b) were quantified and plotted. (d) Experiments similar to thoseshownin(b)wereper- formed, except the concentration of RuvA is varied and the DnaB incubation time is held constant at 2 min. Data from native gel analyses were quantified and plotted as a function of RuvA concentration (open circles). The experiment was then conducted using RuvB in place of DnaB (filled squares). indicated in Figure 7(a). (The faint band that (Figure 7(d)). Low concentrations of RuvA activate accumulates between these products at later time- RuvB-catalyzed branch migration (Figure 7(d), points corresponds to the *1-2 duplex that results filled squares). Half-maximal stimulation of RuvB from unwound strand 1 reannealing with unwound activity occurs at approximately 3 nM RuvA. Half- strand 2.29) maximal inhibition of single-DnaB ring activity To determine if RuvA inhibits DnaB-catalyzed occurs at a similar concentration of RuvA (between unwinding of a heterologous junction, RuvA was 3 nM and 10 nM, Figure 7(d), open circles). Thus, a incubated with the heterologous junction before similar concentration of RuvA that activates RuvB adding DnaB (Figure 7(b)). Only faint products are also inhibits DnaB. This is the concentration range observed for both unwinding (strand *1) and branch in which RuvA binds to the Holliday junction migration (*1-4 duplex). Therefore, in the presence of (Figure 5). Thus, binding of RuvA to the Holliday RuvA, both of these products are produced at a junction at low concentrations likely accounts for slower rate (compare Figure 7(a) with (b)). Quantifi- activation of RuvB and inhibition of DnaB. cation of all products shows that RuvA substantially inhibits DnaB activity on this forked heterologous RuvA inhibits double-DnaB ring-catalyzed Holliday junction (Figure 7(c)). Thus, RuvA inhibits unwinding, but not branch migration both DnaB-catalyzed unwinding and branch migration of the heterologous junction, while RuvA RuvA inhibits branch migration of a heterologous activates RuvB-catalyzed branch migration on this junction catalyzed by a single DnaB ring. Does same substrate (not shown, but similar to the result in RuvA inhibit branch migration of a heterologous Figure 6(e)). junction catalyzed by two opposing DnaB rings? Next, we examined the concentration-depen- RuvA loads two opposing RuvB rings onto junction dence of RuvA for inhibition of DnaB, and for DNA; thus, the effect of RuvA on two opposing activation of RuvB. Experiments were performed as DnaB rings may provide further insight into RuvA described above, except the concentration of RuvA mechanism during RuvAB function. Recombi- was varied and the time was held constant nation processes must sometimes occur over RuvA Promotes Branch Migration 483 regions of unmatched sequences, such as during (Figure 8(a), scheme (ii) and gel). Free strand *1 DNA damage, and it is therefore relevant to test the increases in intensity for the first 4 min of the mobility of RuvA over heterologous regions. Others reaction as the *1,4 duplex is unwound by DnaB have demonstrated that RuvAB can catalyze branch (Figure 8(a), filled squares in graph). The levels of migration over regions of DNA heterology,34,36,38 *1,4 duplex are roughly constant in the first 4 min but the role of RuvA has not been distinguished of the reaction (Figure 8(a), open circles in from that of RuvB in these publications. Below, we graph). This result is explained if the rate of once again use DnaB in conjunction with RuvA to branch migration, shown in scheme (i), is similar study the role of RuvA in heterologous branch to the rate of secondary unwinding, shown in migration. scheme (ii). DnaB catalyzes branch migration of a long To determine if RuvA blocks this reaction, the heterologous junction if two opposing DnaB rings substrate was incubated with RuvA before adding are loaded onto the substrate.35 To load two DnaB (Figure 8(b)). The opposing DnaB rings DnaB rings onto the DNA, a long heterologous catalyze branch migration of this substrate rapidly, junction was constructed that contains two 50 tails even in the presence of RuvA, producing the *1-4 on opposite sides of the junction (Figure 8(a)). duplex product (Figure 8(b), scheme (i) and gel). DnaB rapidly catalyzes branch migration of this The concentration of *1-4 duplex continues to substrate, as expected, producing the *1,4 duplex increase during the first 4 min of the reaction product (Figure 7(a), scheme (i), and gel). There (Figure 8(b), open circles in the graph). In fact, the is a secondary reaction, where the *1-4 duplex is levels of branch migration product rise higher in the unwound by DnaB to yield free strand *1 presence of RuvA than in the absence of RuvA

Figure 8. RuvA inhibits double- DnaB ring-catalyzed unwinding, but not branch migration. The model above each gel is used to orient the reader, and was determined from the experimental gel evidence below. (a) DnaB acts on a long heterologous Holliday junction with two 50 tails. The 1-2 and 3-4 duplexes are 45 bp in length, and the 1-4 and 2-3 duplexes are 25 bp in length. Each 50 tail is composed of 30 dT. Oligonucleo- tides used to form this substrate are provided in Table 1. DnaB was incubated with the junction illustrated for the periods of time indicated. Native gel analysis of the reaction is shown. The arrows next to the gel show the migration distance of DNA products as determined by radiolabeled DNA markers electrophoresed in the same gel. Markers are cropped from the gel images for clarity. The accumulation of the *1,4 duplex (open circles) and free strand *1 (filled squares) are quantified and plotted. (b) Same as (a), except the substrate is incubated with RuvA for 1 min before adding DnaB. (c) Total product accumulation from gels shown in (a) (open diamonds) and (b) (filled circles) were quantified and plotted. (d) Experiments similar to those shown in (b) were performed, except the concentration of RuvA is varied and the DnaB incubation time is held constant at 2 min. Data from native gel analyses were quantified and plotted as a function of RuvA concentration (filled triangles). A similar experiment was conducted using a long heterologous Holliday junction with two forks. The concentration of RuvA was varied and the DnaB incubation time is held constant at 30 s (open circles). DnaB primarily catalyzes unwinding of this two-forked substrate (not shown). DnaB incubation times were chosen to roughly match product accumulation in the absence of RuvA. 484 RuvA Promotes Branch Migration

(compare open circles in the graphs in Figure 8(a) The ability of RuvA at high concentrations to and (b)). This may be due to RuvA inhibiting the bind to a free forked-duplex structure and slightly secondary reaction of *1-4 duplex unwinding as block DnaB activity, as demonstrated in Figure 9, the accumulation of free strand *1 is much slower in raises the question of why RuvA does not bind to the presence of RuvA (see scheme (ii) and compare forked DNA structures when they are part of filled squares in the graphs in Figure 8(a) and (b)). junction DNA in other experiments in this study To test if RuvA inhibits the unwinding reaction of (Figures 3 and 7), thereby partially inhibiting DnaB scheme (ii) in Figure 8(b), the *1-4 duplex was activity. However, the single-stranded tail regions incubated with DnaB in the absence and in the of strands 1 and 4 in the forked-duplex of Figure 9 presence of 300 nM RuvA, the same concentration are not homopolymeric, and thus may form of RuvA as that used in Figure 8(b) (Figure 9). This secondary structures to which RuvA has some high concentration of RuvA slightly inhibits DnaB- affinity. DnaB can unwind this substrate because catalyzed unwinding of this duplex. Thus, RuvA strand 4 retains enough single-stranded character partially blocks reaction (ii) in Figure 8(b), explain- for DnaB-loading. In contrast, the single-stranded ing why in the presence of RuvA, free strand *1 regions of the substrates in Figures 3 and 7 are levels are low and *1-4 duplex product accumulates composed entirely of dT. These sequences form no substantially. secondary structure, and RuvA cannot bind to them The rate of accumulation of all products in (data not shown). Thus, RuvA inhibition of Figure 8(a) and (b) is plotted in the graph shown unwinding in Figures 3 and 7 is presumed to be in Figure 8(c). RuvA does not inhibit the rate of the result of RuvA binding to the DNA branch- accumulation of all products. All products in point. Figure 8(a) and (b) are derived from branch RuvA inhibits single-DnaB ring unwinding and migration of the junction substrate (reaction (i)).29 branch migration activity of a heterologous junc- Thus, RuvA inhibits DnaB unwinding of the *1-4 tion, but it does not inhibit double-ring branch duplex, but it does not inhibit DnaB branch migration activity. We next investigated if RuvA migration of the heterologous junction substrate inhibits double-DnaB ring unwinding activity. We with two 50 tails. This observation indicates that two incubated DnaB with a heterologous junction with DnaB pumps overcome RuvA inhibition of branch forks positioned on either side of the junction migration of a heterologous junction. (Figure 8(d)). DnaB primarily catalyzes unwinding of this substrate (not shown). After 1 min incubation with RuvA, DnaB was added for 30 s. The concentration of RuvA was varied from 0 nM to 300 nM. Quantification of the reaction is shown in Figure 8(d) (open circles), and compared to a similar experiment using the substrate in Figure 8(b) (filled triangles, 2 min DnaB). The result shows that RuvA still inhibits double-DnaB activity on the unwinding substrate, but not the branch-migration substrate. We have shown elsewhere that two DnaB rings work in conjunction at this heterologous junction to power branch migration. The combined power of the two DnaB pumps likely also push RuvA over non-complementary base-pairs during branch migration (Figure 10(d)). However, the need to dislodge RuvA presumably underlies inhibition of helicase activity, even when two DnaB rings are present. Although we cannot rule out the possibility that RuvA is dislodged during branch migration over DNA heterology (Figure 8(d), filled triangles), it is unlikely, given the weak ability of two DnaB pumps to dislodge RuvA during unwinding (Figure 8(d), open circles).

Discussion Figure 9. RuvA inhibits DnaB-catalyzed unwinding of forked-duplex DNA. (a) DnaB unwinds forked-duplex RuvA blocks unwinding, but not branch DNA. The substrate is the primary branch migration migration product of Figure 8(a) and (b). Oligonucleotides used to form this substrate are provided in Table 1. DnaB was incubated with the junction illustrated for the time RuvA binds Holliday junction DNA tightly indicated. Native gel analysis of the reaction is shown. in vitro (Figure 5). Once bound to a Holliday (b) Same as (a), except the substrate is pre-incubated with junction, RuvA inhibits DnaB unwinding activity RuvA for 1 min prior to adding DnaB. (Figures 2(f) and 7(c)). Even when two DnaB RuvA Promotes Branch Migration 485

Figure 10. Mobilization of the RuvA sliding collar at a Holliday junction by either one or two DNA pumps. (a) RuvA inhibits DNA unwinding at a Holliday junction. If the junction were to be unwound by a helicase, the Holliday junction will be destroyed and RuvA will likely be displaced. Thus, tight binding of the RuvA collar to the Holliday junction blocks unwinding by DnaB when it encircles one DNA strand. (b) RuvA does not inhibit homologous branch migration catalyzed by a single duplex DNA pump like DnaB encircling two DNA strands. During branch migration of a homologous junction, the junction structure is preserved as it translocates. The RuvA collar may slide along with the junction at little energetic cost. Thus, the RuvA sliding collar is readily mobilized for branch migration at a homologous junction, and does not require specific protein activation from RuvB. (c) During branch migration of a heterologous junction, two of the duplex arms will change from annealed duplex to unannealed duplex. The RuvA collar is optimized for binding to fully annealed duplex arms; thus, RuvA junction sliding is inhibited by unannealed DNA. (d) Two opposing DNA pumps mobilize the RuvA sliding collar at a heterologous junction, overcoming resistance of RuvA sliding over unannealed DNA. The RuvA sliding collar works in conjunction with two opposing RuvB rings in vivo. These two RuvB rings may enable the RuvA collar to slide over regions of DNA heterology or DNA lesions. hexamers act on opposite sides of the RuvA- recognizes whether DnaB is surrounding one DNA junction, RuvA continues to block DnaB-catalyzed strand or two. RuvA likely evolved this strand unwinding activity (Figure 8(d)). Thus, the RuvA- specificity to help ensure that RuvA is mobile only junction is quite stable and resistant to DNA in the correct context of RuvB-catalyzed branch remodeling activity that involves unwinding. In migration. RuvB usually surrounds two strands contrast, RuvA does not block DnaB-catalyzed in vivo, and RuvA does not block RuvB when RuvB branch migration activity on a homologous Holli- surrounds two strands to power branch migration. day junction (Figure 2(c)). At a heterologous However, if RuvB were to mistakenly surround one junction, RuvA blocks single-DnaB ring branch strand, RuvA would probably block RuvB action migration activity (Figure 6(c)), but not double just as it blocks DnaB action. RuvB may mistakenly ring activity (Figure 8(d)). Thus, RuvA blocks surround one DNA strand during extensive DNA unwinding activity but not branch migration damage with unannealed DNA strands. RuvB has activity. This result is supported further by the been shown to act as a DNA helicase in vitro when experiment illustrated by Figure 2, in which RuvA surrounding one DNA strand,31 and RuvA may blocks DnaB-catalyzed unwinding, but not branch block this aberrant function at a Holliday junction migration, at the same junction. in vivo. RuvA may block other helicases at a At a homologous junction, RuvA blocks DnaB Holliday junction by this mechanism as well. activity when the helicase surrounds one strand, The data presented here suggest that RuvA slides but RuvA does not block DnaB when the hexameric along homologous junction DNA during branch ring surrounds two strands (Figures 2 and 3). Thus, migration, but not unwinding. Why does RuvA RuvA is highly specific in its inhibition, as RuvA not slide along the Holliday junction during 486 RuvA Promotes Branch Migration unwinding? In other words, how can RuvA reaction (Figure 10(a)). In contrast, during DnaB- distinguish whether DnaB is surrounding one or catalyzed branch migration of an RuvA-homo- two DNA strands? Our previous studies of DnaB logous junction complex, the fully annealed junc- suggest that the ring-shaped protein functions by a tion structure is continuously preserved, and thus similar mechanism, whether the helicase ring RuvA can stay bound throughout the process surrounds one strand during unwinding, or two (Figure 10(b)). The RuvA collar can simply slide DNA strands during branch migration. In either along all four duplex arms of the junction while case, DnaB primarily pumps one DNA strand DnaB drives branch migration. Thus, RuvA binds through the central channel. The primary difference Holliday junctions stably, and it is difficult to between the two modes of action is that, during dislodge this protein completely from the DNA unwinding, the strands become separated and one substrate. However, RuvA may simply slide along strand passes outside of the DnaB ring, whereas the junction during branch migration. The homo- during branch migration, both strands pass through logous junction used here bears a slight degree of the DnaB ring. heterology (five bases); thus, RuvA can tolerate a While it is unclear at present how RuvA can small degree of heterology and still slide freely. discriminate between the two modes of DnaB There is no evidence that DnaB and RuvA function action, this puzzle may have a basis similar to together in vivo. Thus, the observation that RuvA is that of the replication termination system in readily mobilized by DnaB for branch migration bacteria, the Tus–Ter complex. The Tus–Ter suggests that RuvA has an intrinsic capacity for complex blocks DnaB when the helicase movement on DNA during branch migration. approaches from one direction (the permissive Moreover, our results show that RuvA does not direction), but not the other (the non-permissive require specific contacts with its in vivo protein direction).39 The Tus–Ter system is remarkably partner, RuvB, to move readily on DNA branch similar to the findings here for RuvA, in that, migration. even for helicases that the Tus protein does not It has been proposed that RuvA binds a Holliday interact with in vivo, Tus blocks helicase action junction tightly and, upon interaction with RuvB, from only one direction.40,41 Although this RuvA is transformed to a weak DNA binder, mechanism is not understood completely, an capable of movement during branch migration.10 interesting model has been proposed by the Evidence presented here suggests that RuvA binds Dixon laboratory.39 In this model, the ssDNA to Holliday junctions tightly but yet is freely mobile produced by unwinding as DnaB approaches in the direction of branch migration, without the adheres to the ssDNA pocket in Tus, holding it requirement for activation by RuvB. Why does tight to DNA and preventing DnaB from RuvA have an intrinsic capacity to slide in the displacing it. Since Tus is asymmetric, the direction of branch migration? RuvA needs to slide ssDNA pocket faces the duplex in one direction rapidly when functioning with RuvB during branch but not the other. Hence, DnaB approaching from migration. Since RuvA has evolved to slide freely the opposite side can displace Tus, since the during branch migration, all of the energy of the ssDNA produced by the helicase does not RuvB motor can be utilized for branch migration. interact with the ssDNA pocket on the other Thus, RuvA does not impede the function of its side of Tus. Perhaps RuvA acts similarly and in vivo partner, RuvB. Furthermore, since RuvA binds ssDNA, allowing it to grip and not slide remains bound tightly to Holliday junction DNA (RuvA is symmetric, and the hypothetical ssDNA during branch migration, RuvA can continue to pocket would be seen by all directions). This function as a sliding collar to preserve Holliday ssDNA is produced only when DnaB acts as a junction structure, and ultimately aid in the helicase, allowing RuvA to bind tightly to the recruitment of the RuvC resolvase. Finally, this DNA side and not slide. But when DnaB work suggests that RuvA is part of a family of encirclesduplexDNA,thessDNAisnot nucleic acid sliding proteins that grip DNA tightly produced and RuvA develops no extra affinity yet are also highly mobile (see below). for DNA and thereby slides as DnaB pushes on it. Ultimately, this speculation may or may not be Comparison of the RuvA collar with other DNA correct, but at least it provides a mechanistic sliding proteins basis for understanding why RuvA slides while DnaB encircles two strands, and does not slide RuvA is mobilized readily during branch when it acts as a helicase. migration, even though it is not displaced easily from DNA. How can RuvA bind tightly to DNA, RuvA is intrinsically able to slide during branch but also be readily pushed to slide along it? In this migration respect, RuvA has some similarity to the polymerase processivity clamp. In E. coli,theb-sliding As described above, RuvA likely slides during clamp confers processivity to the replicative poly- branch migration, but not during unwinding. For merase.42 During replication, the b-clamp remains DnaB to catalyze unwinding of an RuvA-bound bound to the DNA duplex, sliding along the DNA Holliday junction, RuvA must be displaced as the at a rate of 500–1000 bp/s. However, it is difficult to four-way junction structure is destroyed during the dislodge the b-clamp from the DNA, and an RuvA Promotes Branch Migration 487 accessory protein is required to unload the RuvA slides over a heterologous junction with b-clamp.43 two opposing DNA pumps The b-clamp forms a ring around the DNA, which enables tight DNA binding while retaining Recombination processes must sometimes occur mobility to slide along DNA.44,45 Similar to the over regions of unmatched sequences, such as b-clamp, RuvA binds DNA topologically by during DNA damage, and it is therefore important forming a collar or sandwich structure around to understand the mobility of RuvA over hetero- all four arms of the Holliday junction.46 Thus, the logous regions. It has been shown that RuvAB topological binding of RuvA at a Holliday catalyzes branch migration over regions of DNA junction ensures that RuvA does not dissociate heterology with less efficiency compared to homo- readily from DNA during branch migration, logous DNA,34,36,38 but the role of RuvA in this while still allowing mobility on the DNA arms process was not dissected from RuvB in these by sliding. However, there are important differ- earlier studies. We show in this study that if the ences between the b-clamp and the RuvA collar. junction is homologous, RuvA is freely mobile to While the b-clamp surrounds one DNA duplex, slide over the junction, as discussed above. RuvA is a four-way collar that encircles all four However, we show also that if the junction is duplex arms simultaneously. Thus, for RuvA, all heterologous, the unannealed strands within the four duplexes must slide within it at the same RuvA collar inhibit RuvA sliding. DnaB cannot time (Figure 10(b)). Moreover, the b-dimer ring is mobilize RuvA at a heterologous junction if the a more stable multimer than the RuvA-octamer protein is loaded on one junction arm. However, if collar,47 explaining why the b-dimer requires an DnaB is loaded onto opposing junction arms, RuvA accessory protein to crack open the dimer to load no longer inhibits DnaB-catalyzed branch around DNA, while the RuvA collar can migration. Thus, two opposing DNA pumps can assemble spontaneously around DNA from two overcome the inhibition to RuvA sliding that DNA tetramers. heterology creates. RuvA normally functions with The crystal structure of the b-clamp bound to opposing RuvB rings. The two opposing RuvB rings DNA has not been solved, but the structure of may enable the RuvA collar to slide over regions of the b-clamp alone reveals conserved positive and heterology, consistent with the published mobility polar residues within the central channel.44 These of the RuvAB complex over regions of hetero- residues may make direct or water-mediated logy.34,36,38 Thus, in vivo, two RuvB pumps may contact with the DNA duplex, further ensuring function in coordination to mobilize RuvA through that the protein remains bound to DNA while regions of DNA heterology or DNA lesions. sliding for millions of base-pairs during genome replication. Although RuvA–DNA crystal struc- RuvA may function in vivo to stabilize Holliday tures have not been solved at sufficiently high junction structure and limit action to branch resolution to map protein–DNA interactions migration unambiguously, RuvA has conserved positive and polar residues that may make direct and In vivo, a Holliday junction arises after water-mediated contact with Holliday junction RecA-mediated DNA recombination. The Holli- DNA.10 Water-mediated hydrogen bonds are day junction structure must be processed to likely to be quite mobile in an aqueous environ- linear duplexes to restore genomic integrity. The ment.Thus,thenatureofchemicalcontact RuvABC proteins are responsible for this import- between RuvA or the b-clamp and DNA appears ant cellular function, and RuvA is the first of designed for DNA sliding. Moreover, RuvA these three proteins to bind the Holliday junction. primarily contacts the phosphate backbone of We demonstrate here that RuvA, acting alone, the DNA, which will maintain a uniform inhibits DnaB unwinding activity substantially by structure as RuvA slides along the Holliday binding tightly to a Holliday junction. junction. However, the b-clamp may have fewer The collision of a replication fork with an direct contacts with DNA than RuvA, explaining RuvA-bound Holliday junction may not be a how the b-clamp may slide more freely along frequent event. However, the RuvA collar may DNA than RuvA. In addition, the RuvA sliding function in vivo to protect the Holliday junction collar may not slide on its own, but may need a from the deleterious action of other DNA- push by a duplex DNA pump for sliding motion. metabolizing enzymes until RuvB is recruited to Other DNA-metabolizing enzymes, such as the Holliday junction. RuvA may thus protect a restriction enzymes, have been proposed to slide Holliday junction from helicases, nucleases, and along DNA.48,49 Thus, protein sliding along DNA other enzymes that would destroy the Holliday in a non-sequence-specific manner may be used junction structure. Furthermore, once RuvB is by proteins to accomplish a variety of functions bound to RuvA and branch migration ensues, the in the cell. Many nucleic acid-binding proteins RuvA can work as a sliding collar to allow must traverse along DNA at a very fast rate to efficient RuvB-catalyzed branch migration while accomplish their function, and DNA sliding is an preserving the structure of the Holliday junction elegant mechanism for proteins to move quickly from the deleterious effect of other DNA meta- along DNA without dissociation. bolizing enzymes. 488 RuvA Promotes Branch Migration

Materials and Methods unless stated otherwise. RuvA was incubated with substrate DNA for 1 min at 37 8C before addition of RuvB or DnaB, and then incubated further as indicated Proteins and DNA in the Figure or described in the Figure legend. Reactions were quenched upon adding 1 mlof Proteins were expressed in E. coli and puriﬁed as 50 51 proteinase K (10 mg/ml), and incubated at 37 8C for an described: RuvA, RuvB, and DnaB. DNA oligonucleo- additional 1 min, followed by addition of 5 mlof2% tides used to construct the substrates in this work are 24 (w/v) SDS, 80 mM EDTA. For gel analysis, 5 ml of 15% given in Table 1, and were synthesized as described. (v/v) Ficoll (type 400; Pharmacia) and 0.25% (w/v) Oligonucleotide strands were labeled with 32P at the 0 24 xylene cyanol FF were added. Samples were snap- 5 -end as described. frozen in a dry ice/ethanol bath and stored at K20 8C. Before gel electrophoresis, each reaction tube was Branch migration assays incubated at room temperature in a waterbath for 4 min to reduce intrastrand base-pairing. All manipulations were performed in microfuge For some experiments, the rate of spontaneous DNA tubes on ice, and then shifted to 37 8C, unless stated reannealing is fast relative to the time-course of the otherwise. Oligonucleotides were annealed to form experiment. For these reactions, unlabeled oligonucleotides DNA substrates as described.35 Enzyme reactions were added in tenfold excess to inhibit spontaneous DNA were incubated at 37 8Cforthelengthsoftime reannealing. The DNA traps used in each experiment are indicated, and contained 1 nM DNA substrate (concen- listed in Table 1. Unlabeled DNA trap was added tration of labeled strand) in 20 mM Tris–HCl (pH 7.5) immediately before protein addition. High concentrations 5 mM ATP, 5 mM creatine phosphate, 20 mg/ml of of DNA (O500 nM) are required to fully trap DnaB in these creatine kinase, 10 mM magnesium acetate, 20% (v/v) reactions. At these high concentrations, the DNA trap glycerol, 100 mM EDTA, 40 mg/ml of bovine serum inhibits DnaB activity at the Holliday junction. Thus, we do albumin, 5 mM DTT, in a ﬁnal volume of 10 ml. Protein not fully trap DnaB in this study, but we do use DNA at concentrations were 500 nM DnaB (hexamer), 300 nM lower concentrations to trap DNA products generated RuvA (monomer), and/or 1 mM RuvB (monomer), during the reaction to prevent DNA reannealing.

Table 1. Sequences of DNA oligonucleotides

Figure Oligonucleotides used Cold DNA trap 1(a) 1TE, 2E, 3O, and 4O 1(b) 1T, 2, 3, and 4 2(a) and (b) 1TE, 2E, 3O, and 4O 1TE-4O 2(d) and (e) 1TE, 2E, 3O, 4ODS, 5DS 2E 3(a) and (b) 1TE, 2E, 3O, and 4OT 2E-3O, and 2E 4(a) and (d) 1E, 2E, 3E, and 4E 1E-2E, and 1E-4E 5(b) 1, 2, 3, and 4 None 6(a) and (b) 1T, 2, 3, and 4 1T-4 6(e) and (f) 1T, 2, 3, and 4 1T-4, and 1T-2 7(a) and (b) 1T, 2, 3, and 4T 1T-4T, and 1T 8(a) and (b) 1TE, 2E, 3TE, and 4E 1TE-4E 8(d) 1TE, 2E, 3TE, and 4E 1TE-4E 1TE, 2TE, 3TE, and 4TE 1TE-4TE 9(a) and (b) 1TE and 4E None 1 50-GAC GCT GCC GAA TTC TGG CTT GCT AGG ACA TCT TTG CCC ACG TTG ACC CG-30 1E 50-GAC GCT GCC GAA TTC TGG CTT GCT AGG ACA TCT TTG CCC ACG TTG ACC CGA TCG CTT AGG TAC GTT AAC C-30 1T 50-TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT GAC GCT GCC GAA TTC TGG CTT GCT AGG ACA TCT TTG CCC ACG TTG ACC CG-30 1TE 50-TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT GAC GCT GCC GAA TTC TGG CTT GCT AGG ACA TCT TTG CCC ACG TTG ACC CGA TCG CTT AGG TAC GTT AAC C-30 2 50-CGG GTC AAC GTG GGC AAA GAT GTC CTA GCA ATG TAA TCG TCT ATG ACG TC-30 2E 50-GGT TAA CG T ACC TAA GCG ATC GGG TCA ACG TGG GCA AAG ATG TCC TAG CAA TGT AAT CGT CTA TGA CGT C-30 2TE 50-GGT TAA CG T ACC TAA GCG ATC GGG TCA ACG TGG GCA AAG ATG TCC TAG CAA TGT AAT CGT CTA TGA CGT C TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT-30 3 50-GAC GTC ATA GAC GAT TAC ATT GCT AGG ACA TGC TGT CTA GAG ACT ATC GC-30 3E 50-GAC GTC ATA GAC GAT TAC ATT GCT AGG ACA TGC TGT CTA GAG ACT ATC GCA CGC TTT CGA ACG AGT CTT A-30 3O 50-GAC GTC ATA GAC GAT TAC ATT GCT AGG ACA TGC TGT CTC ACG TTG ACC CGA TCG CTT AGG TAC GTT AAC C-30 3TE 50-TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT GAC GTC ATA GAC GAT TAC ATT GCT AGG ACA TGC TGT CTA GAG ACT ATC GCA CGC TTT CGA ACG AGT CTT A-30 4 50-GCG ATA GTC TCT AGA CAG CAT GTC CTA GCA AGC CAG AAT TCG GCA GCG TC-30 4E 50-TAA GAC TCG TTC GAA AGC GTG CGA TAG TCT CTA GAC AGC ATG TCC TAG CAA GCC AGA ATT CGG CAG CGT C-30 4O 50-GGT TAA CGT ACC TAA GCG ATC GGG TCA ACG TGA GAC AGC ATG TCC TAG CAA GCC AGA ATT CGG CAG CGT C-30 4ODS 50-GGT TAA CGT ACC TAA GCG ATC GGG TCA ACG TGA GAC AGC ATG TCC TAG CAA GCC AGA ATT CGG CAG CGT C TTT TCG ATA TCC ATC CAT CCA TCC ATG CAC-30 4OT 50-GGT TAA CGT ACC TAA GCG ATC GGG TCA ACG TGA GAC AGC ATG TCC TAG CAA GCC AGA ATT CGG CAG CGT C TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT-30 4TE 50-TAA GAC TCG TTC GAA AGC GTG CGA TAG TCT CTA GAC AGC ATG TCC TAG CAA GCC AGA ATT CGG CAG CGT C TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT-30 5DS 50-GTG CAT GGA TGG ATG GAT GGA TAT CGA-30 RuvA Promotes Branch Migration 489

DNA products were separated from DNA substrate in 8. Ingleston, S. M., Sharples, G. J. & Lloyd, R. G. (2000). a native polyacrylamide gel as described.29 The gels were The acidic pin of RuvA modulates Holliday junction dried, exposed to a Phosphor-imaging screen, and binding and processing by the RuvABC resolvasome. quantiﬁed as described.29 The arrows next to the gel EMBO J. 19, 6266–6274. show the migration distance of DNA products as 9. Hargreaves, D., Rice, D. W., Sedelnikova, S. E., determined by radiolabeled DNA markers in the same Artymiuk, P. J., Lloyd, R. G. & Rafferty, J. B. (1998). gel. Markers are cropped from the gel images for clarity. Crystal structure of E. coli RuvA with bound DNA Holliday junction at 6 A˚ resolution. Nature Struct. Biol. Electrophoretic mobility-shift assay 5, 441–446. 10. Ariyoshi, M., Nishino, T., Iwasaki, H., Shinagawa, H. RuvA was incubated with radiolabeled junction DNA & Morikawa, K. (2000). Crystal structure of the for 1 min at 37 8C under conditions identical with those Holliday junction DNA in complex with a single used for branch migration assays. Then 5 ml of 15% Ficoll RuvA tetramer. Proc. Natl Acad. Sci. USA, 97, (type 400; Pharmacia) and 0.25% xylene cyanol FF was 8257–8262. added to the sample. Protein–DNA complexes were 11. Yamada, K., Miyata, T., Tsuchiya, D., Oyama, T., separated from free DNA complexes by native gel Fujiwara, Y., Ohnishi, T. et al. (2002). Crystal structure electrophoresis. The gel was composed of 4% (w/v) of the RuvA–RuvB complex: a structural basis for the polyacrylamide (29:1 (w/w) acrylamide/bis acrylamide) Holliday junction migrating motor machinery. Mol. in a buffer of TBE (90 mM Tris–HCl/borate, 1 mM EDTA, Cell, 10, 671–681. pH 8.0). The running buffer was TBE. The gel was 12. Yamada, K., Kunishima, N., Mayanagi, K., Ohnishi, T., developed at 150 V at room temperature until the free Nishino, T., Iwasaki, H. et al. (2001). Crystal structure of DNA was near the bottom of the gel. The gels were dried, the Holliday junction migration motor protein RuvB exposed to a Phosphor-imaging screen, and quantiﬁed as from Thermus thermophilus HB8. Proc. Natl Acad. Sci. described.29 USA, 98, 1442–1447. 13. Putnam, C. D., Clancy, S. B., Tsuruta, H., Gonzalez, S., Wetmur, J. G. & Tainer, J. A. (2001). Structure and mechanism of the RuvB Holliday junction branch migration motor. J. Mol. Biol. 311, 297–310. Acknowledgements 14. Parsons, C. A., Tsaneva, I., Lloyd, R. G. & West, S. C. (1992). Interaction of Escherichia coli RuvA and RuvB We thank Nicholas Dixon, Taekjip Ha, and Nancy proteins with synthetic Holliday junctions. Proc. Natl Horton for useful discussions regarding this work. Acad. Sci. USA, 89, 5452–5456. 15. Hiom, K. & West, S. C. (1995). Branch migration We thank Steve West and Ken Marians for during homologous recombination: assembly of a providing expression vectors for the Ruv proteins. RuvAB Holliday junction complex in vitro. Cell, 80, Thanks to everyone in the O’Donnell laboratory. 787–793. This research was supported by grant GM38839 16. Muller, B., Tsaneva, I. R. & West, S. C. (1993). Branch from the NIH and by HHMI. D.L.K. was the Leon migration of Holliday junctions promoted by the and Toby Cooperman Fellow of the Damon Runyon Escherichia coli RuvA and RuvB proteins. II. Inter- Cancer Research Foundation (DRG # 1663). action of RuvB with DNA. J. Biol. Chem. 268, 17185–17189. 17. Patel, S. S. & Picha, K. M. (2000). Structure and References function of hexameric helicases. Annu. Rev. Biochem. 69, 651–697. 1. Lusetti, S. L. & Cox, M. M. (2002). The bacterial RecA 18. Sawaya, M. R., Guo, S., Tabor, S., Richardson, C. C. & protein and the recombinational DNA repair of Ellenberger, T. (1999). Crystal structure of the helicase stalled replication forks. Annu. Rev. Biochem. 71, domain from the replicative helicase-primase of 71–100. bacteriophage T7. Cell, 99, 167–177. 2. West, S. C. (1997). Processing of recombination 19. Singleton, M. R., Sawaya, M. R., Ellenberger, T. & intermediates by the RuvABC proteins. Annu. Rev. Wigley, D. B. (2000). Crystal structure of T7 gene 4 Genet. 31, 213–244. ring helicase indicates a mechanism for sequential 3. Parsons, C. A., Stasiak, A., Bennett, R. J. & West, S. C. hydrolysis of nucleotides. Cell, 101, 589–600. (1995). Structure of a multisubunit complex that 20. Baker, T. A., Sekimizu, K., Funnell, B. E. & Kornberg, A. promotes DNA branch migration. Nature, 374, (1986). Extensive unwinding of the plasmid template 375–378. during staged enzymatic initiation of DNA replication 4. Yu, X., West, S. C. & Egelman, E. H. (1997). Structure from the origin of the Escherichia coli chromosome. Cell, and subunit composition of the RuvAB-Holliday 45, 53–64. junction complex. J. Mol. Biol. 266, 217–222. 21. Bujalowski, W., Klonowska, M. M. & Jezewska, M. J. 5. Tsaneva, I. R., Muller, B. & West, S. C. (1992). ATP- (1994). Oligomeric structure of Escherichia coli primary dependent branch migration of Holliday junctions replicative helicase DnaB protein. J. Biol. Chem. 269, promoted by the RuvA and RuvB proteins of E. coli. 31350–31358. Cell, 69, 1171–1180. 22. Yang, S., Yu, X., VanLoock, M., Jezewska, M., 6. Eggleston, A. K., Mitchell, A. H. & West, S. C. (1997). Bujalowski, W. & Egelman, E. (2002). Flexibility of the In vitro reconstitution of the late steps of genetic rings: structural asymmetry in the DnaB hexameric recombination in E. coli. Cell, 89, 607–617. helicase. J. Mol. Biol. 321, 839–849. 7. Eggleston, A. K. & West, S. C. (2000). Cleavage of 23. Jezewska, M. J., Rajendran, S., Bujalowska, D. & Holliday junctions by the Escherichia coli RivABC Bujalowski, W. (1998). Does single-stranded DNA complex. J. Biol. Chem. 275, 26467–26476. pass through the inner channel of the protein hexamer 490 RuvA Promotes Branch Migration

in complex with the Escherichia coli DnaB helicase? 38. Dennis, C., Fedorov, A., Kas, E., Salome, L. & Fluorescence energy transfer studies. J. Biol. Chem. Grigoriev, M. (2004). RuvAB-directed branch 273, 10515–10529. migration of individual Holliday junctions is 24. Kaplan, D. L. (2000). The 30-tail of a forked-duplex impeded by sequence heterology. EMBO J. 23, sterically determines whether one or two DNA 2413–2422. strands pass through the central channel of a 39. Mulugu, S., Shamsuzzaman, A. P. A., Taylor, J., replication-fork helicase. J. Mol. Biol. 301, 285–299. Alexander, K. & Bastia, D. (2001). Mechanism of 25. Jezewska, M. J., Rajendran, S. & Bujalowski, W. (1998). termination of DNA replication of Escherichia coli Complex of Escherichia coli primary replicative heli- involves helicase–contrahelicase interaction. Proc. case DnaB protein with a replication fork: recognition Natl Acad. Sci. USA, 98, 9569–9574. and structure. Biochemistry, 37, 3116–3136. 40. Lee, E. H., Kornberg, A., Hidaka, M., Kobayashi, T. & 26. Delagoutte, E. & von Hippel, P. H. (2003). Helicase Horiuchi, T. (1989). Escherichia coli replication termin- mechanisms and the coupling of helicases within ation protein impedes the action of helicases. Proc. macromolecular machines. Part II: integration of Natl Acad. Sci USA, 86, 9104–9108. helicases into cellular processes. Quart. Rev. Biophys. 41. Bedrosian, C. L. & Bastia, D. (1991). Escherichia coli 36, 1–69. replication terminator protein impedes simian virus 27. Richardson, R. W. & Nossal, N. G. (1989). Characteri- 40 (SV40) DNA replication fork movement and SV40 zation of the bacteriophage T4 gene 41 DNA helicase. large tumor antigen helicase activity in vitro at a J. Biol. Chem. 264, 4725–4731. prokaryotic terminus sequence. Proc. Natl Acad. Sci. 28. Cunningham, E. L. & Berger, J. M. (2005). Unraveling USA, 88, 2618–2622. the early steps of prokaryotic replication. Curr. Opin. 42. Kelman, Z. & O’Donnell, M. (1995). DNA polymerase Struct. Biol. 15, 68–76. III holoenzyme: structure and function of a chromo- 29. Kaplan, D. L. & O’Donnell, M. (2002). DnaB drives somal replicating machine. Annu. Rev. Biochem. 64, DNA branch migration and dislodges proteins while 171–200. encircling two DNA strands. Mol. Cell, 10, 647–657. 43. Leu, F. P., Hingorani, M. M., Turner, J. & O’Donnell, M. 30. Rajendran, S., Jezewska, M. J. & Bujalowski, W. (2000). (2000). The delta subunit of DNA polymerase III Multiple-step kinetic mechanism of DNA-indepen- holoenzyme serves as a sliding clamp unloader in dent ATP binding and hydrolysis by Escherichia coli Escherichia coli. J. Biol. Chem. 275, 34609–34618. replicative helicase DnaB protein: quantitative anal- 44. Kong, X. P., Onrust, R. O., O’Donnell, M. & Kuriyan, J. ysis using the rapid quench-flow method. J. Mol. Biol. (1992). Three-dimensional structure of the beta 303, 773–795. subunit of E. coli DNA polymerase III holoenzyme is 31. Tsaneva, I. R., Muller, B. & West, S. C. (1993). RuvA a sliding DNA clamp. Cell, 69, 425–437. and RuvB proteins of Escherichia coli exhibit DNA 45. Stukenberg, P. T., Studwell-Vaughan, P. S. & helicase activity in vitro. Proc. Natl Acad. Sci. USA, 90, O’Donnell, M. (1991). Mechanism of the sliding 1315–1319. beta-clamp of DNA polymerase III holoenzyme. 32. LeBowitz, J. H. & McMacken, R. (1986). The J. Biol. Chem. 266, 11328–11334. Escherichia coli dnaB replication protein is a DNA 46. Roe, S. M., Barlow, T., Brown, T., Oram, M., Keeley, A., helicase. J. Biol. Chem. 261, 4738–4748. Tsaneva, I. R. & Pearl, L. H. (1998). Crystal structure of 33. Grigoriev, M. & Hsieh, P. (1998). Migration of a an octameric RuvA-Holliday junction complex. Mol. Holliday junction through a nucleosome directed by Cell, 2, 361–372. the E. coli RuvAB motor protein. Mol. Cell, 2, 373–381. 47. Lee, Y. C., Flora, R., McCafferty, J. A., Gor, J., 34. Iype, L. E., Wood, E. E., Inman, R. B. & Cox, M. M. Tsaneva, I. R. & Perkins, S. J. (2003). A tetramer- (1994). RuvA and RuvB proteins facilitate the bypass octamer equilibrium in Mycobacterium leprae and of heterolgous DNA insertions during RecA protein- Escherichia coli RuvA by analytical ultracentrifuga- mediated DNA strand exchange. J. Biol. Chem. 269, tion. J. Mol. Biol. 333, 677–682. 24967–24978. 48. Viadiu, H. & Aggarwal, A. K. (2000). Structure of 35. Kaplan, D. L. & O’Donnell, M. (2004). Twin DNA BamHI bound to nonspecific DNA: a model for DNA pumps of a hexameric helicase provide power to sliding. Mol. Cell, 5, 889–895. simultaneously melt two duplexes. Mol. Cell, 15, 49. Kampmann, M. (2004). Obstacle bypass in protein 453–465. motion along DNA by two-dimensional rather than 36. Adams, D. E. & West, S. C. (1996). Bypass of DNA one-dimensional sliding. J. Biol. Chem. 279, heterologies during RuvAB-mediated three and four- 38715–38720. strand branch migration. J. Mol. Biol. 263, 582–596. 50. Tsaneva, I., Illing, G., Lloyd, R. & West, S. (1992). 37. Hishida, T., Iwasaki, H., Han, Y. W., Ohnishi, T. & Purification and properties of the RuvA and RuvB Shinagawa, H. (2003). Uncoupling of the ATPase proteins of Escherichia coli. Mol. Gen Genet. 235, 1–10. activity from the branch migration activity of RuvAB 51. Yuzhakov, A., Turner, J. & O’Donnell, M. (1996). protein complexes containing both wild-type and Replisome assembly reveals the basis for asymmetric ATPase-defective RuvB proteins. Genes Cells, 8, function in leading and lagging strand replication. 721–730. Cell, 86, 877–886.

Edited by R. Ebright

(Received 1 September 2005; received in revised form 25 October 2005; accepted 26 October 2005) Available online 16 November 2005